Isolation and Purification of Extracellular Vesicles from Aspergillus fumigatus

Extracellular vesicles (EVs) are increasingly recognized as key players in fungal biology, mediating intercellular communication, modulating virulence, and shaping host-pathogen interactions. Despite their growing importance, the lack of standardized protocols for EV isolation in fungi has hindered reproducibility and the accurate interpretation of functional data. This chapter presents a comprehensive, optimized protocol for the isolation and characterization of EVs produced by Aspergillus fumigatus under both solid and liquid culture conditions. Culture parameters, including medium composition and growth format, are critically evaluated for their impact on EV yield, molecular composition, and biological significance. The protocol encompasses detailed procedures for fungal cultivation, EV isolation via differential ultracentrifugation, and multiparametric characterization through nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and molecular marker profiling, all aligned with MISEV2023 guidelines. Designed to support both exploratory and high-resolution investigations, this standardized approach robustly analyzes EV-associated biomarkers. The isolated EVs are suitable for a range of downstream applications, including studies of immune modulation, molecular composition, fungal pathogenesis, and intercellular signaling dynamics.